Removal of the translation initiator N-formyl-methionine or methionine from a recombinant protein is often critical for its function and stability. For example, it has been shown that such removal is essential for human hemoglobin, interleukin-2, growth hormones or frog ribonucleases (Adachi et al., 2000; Endo et al., 2001; Busby et al., 1987; Liao et al., 2003; Boix et al., 1996; Varshavsky, 1996). For the preparation of proteins with innate N-terminus, various attempts have been made to remove the N-terminal Met. First, cyanogen bromide (CNBr) was used to cleave Met under extreme acidic conditions, but the method is limited to proteins without any internal Met residue (Boix et al., 1996). Second, a protease-specific oligopeptide was introduced in front of a target protein, which was then removed in vitro by the respective protease, e.g., factor Xa, enterokinase or cathepsin C (Belagaje et al., 1997). Third, the N-terminal Met of a protein was removed in vitro by the aminopeptidase of Aeromonas proteolytica (Notomista et al., 1999; Shapiro et al., 1988). Fourth, a signal peptide was introduced in front of target protein and processed in vivo during secretion, but the yield was low (1˜5 mg per liter of culture) (Huang et al., 1998).
Recently, methionine aminopeptidases (MetAPs) were employed to remove the N-terminal Met in E. coli and yeast. There are two types of MetAP, MetAP I and MetAP II, in eukaryotes. Only MetAP I exists in eubacteria; MetAP II is present in archaea (Li et al., 1995; Tahirov et al., 1998). It has been demonstrated that the genes for these MetAPs are essential for the growth of prokaryotes and eukaryotes (Li et al., 1995; Chang et al., 1989). However, the efficiency of Met removal is limited by the size of the side chain (radius<3.68 Å) of the penultimate residue, the residue next to the N-terminal Met (Ben-Bassat et al., 1987; Hirel et al., 1989; Hwang et al., 1999; Chen et al., 2002). Thus, Met removal for proteins with larger or bulky penultimate residues is inefficient. It is also difficult to remove the N-terminal Met when the penultimate residue is charged, either acidic (Glu or Asp) or basic (Lys, Arg or His).
From the structure of E. coli MetAP and bestatin-based inhibitor complex, it was discovered that four residues (Tyr168, Gln233, Met206 and Glu204) reside in the substrate binding pocket (Lowther et al., 1999; Lowther et al., 2000). The Met329 and Gln356 residues of yeast MetAP, corresponding to Met206 and Gln233 of E. coli MetAP, were replaced with Ala. When tested in vitro, these purified MetAP I variants (M329A, Q356A) had significantly increased catalytic activities for oligopeptides with slightly larger penultimate residues, such as Asn, His and Met, but not with bulky or acidic penultimate residues (Roderick et al., 1993; Walker et al., 1999). Therefore, the need exists for a method to remove N-terminal Met that is more universally applicable, particularly a method that can remove the N-terminal Met from a bulky or acidic penultimate residue.